Abstract
BackgroundRecycling of endosomes is important for trafficking and maintenance of proteins at the neuromuscular junction (NMJ). We have previously shown high expression of the endocytic recycling regulator Eps15 homology domain-containing (EHD)1 proteinin the Torpedo californica electric organ, a model tissue for investigating a cholinergic synapse. In this study, we investigated the localization of EHD1 and its paralogs EHD2, EHD3, and EHD4 in mouse skeletal muscle, and assessed the morphological changes in EHD1−/− NMJs.MethodsLocalization of the candidate NMJ protein EHD1 was assessed by confocal microscopy analysis of whole-mount mouse skeletal muscle fibers after direct gene transfer and immunolabeling. The potential function of EHD1 was assessed by specific force measurement and α-bungarotoxin-based endplate morphology mapping in EHD1−/− mouse skeletal muscle.ResultsEndogenous EHD1 localized to primary synaptic clefts of murine NMJ, and this localization was confirmed by expression of recombinant green fluorescent protein labeled-EHD1 in murine skeletal muscle in vivo. EHD1−/− mouse skeletal muscle had normal histology and NMJ morphology, and normal specific force generation during muscle contraction. The EHD 1–4 proteins showed differential localization in skeletal muscle: EHD2 to muscle vasculature, EHD3 to perisynaptic regions, and EHD4 to perinuclear regions and to primary synaptic clefts, but at lower levels than EHD1. Additionally, specific antibodies raised against mammalian EHD1-4 recognized proteins of the expected mass in the T. californica electric organ. Finally, we found that EHD4 expression was more abundant in EHD1−/− mouse skeletal muscle than in wild-type skeletal muscle.ConclusionEHD1 and EHD4 localize to the primary synaptic clefts of the NMJ. Lack of obvious defects in NMJ structure and muscle function in EHD1−/− muscle may be due to functional compensation by other EHD paralogs.
Highlights
Recycling of endosomes is important for trafficking and maintenance of proteins at the neuromuscular junction (NMJ)
We identified several high-abundance proteins including Eps 15 homology domain-containing 1 (EHD1; Swiss-Prot: Q5E9R3), adducin gamma (ADD3; Swiss-Prot:Q9UEY8), laminin receptor protein 1 (LamR1; Swiss-Prot:Q803F6), chromosome 1 open reading frame 123 (C1orf123; SwissProt: Q9NWV4), transgelin-3 (TAGL3; Swiss-Prot: P37805), and transforming growth factor-β-induced (TGFBI; Swiss-Prot:Q15582), which may play a role in synapse structure and maintenance
Eps15 homology domain-containing 1 protein localizes to the primary synaptic cleft in skeletal muscle Our proteomic data identified Eps homology domain-containing 1 (EHD1) as an abundant protein in the T. californica electric organ [1], suggesting that mammalian EHD1 might be an uncharacterized candidate neuromuscular junction protein
Summary
Recycling of endosomes is important for trafficking and maintenance of proteins at the neuromuscular junction (NMJ). We investigated the localization of EHD1 and its paralogs EHD2, EHD3, and EHD4 in mouse skeletal muscle, and assessed the morphological changes in EHD1−/− NMJs. The neuromuscular junction (NMJ) is a subcellular specialization of the myofiber plasma membrane, with nuclear domains directing synaptic gene expression. We previously identified concordance of the and transforming growth factor-β-induced (TGFBI; Swiss-Prot:Q15582), which may play a role in synapse structure and maintenance. This approach of using the proteomic profile of an amplified model synapse should expedite candidate NMJ protein identification and characterization and help inconstructing a more complete NMJ paradigm. EHD1 was investigated as a peripheral membrane protein that functions in clathrinindependent endocytosis and recycling of receptors at the membrane through the tubular endosomal recycling compartment (ERC) [1,2]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
